Dengue virus, a member of the Flaviviridae family, is a Category A pathogen that causes the most prevalent arthropod-borne viral illnesses in humans. The lack of in depth understanding of molecular mechanisms of viral replication and its interaction with the host cell, limits the development of antiviral strategies. The viral genome is an RNA molecule that plays multiple roles during viral replication. It serves as mRNA for translation, a template for RNA amplification, and substrate for encapsidation. A great deal has been learned in the last ten years about the mechanism of DENV genome replication. In contrast, the processes by which the genome is recruited by the capsid protein during viral particle formation and then released from the nucleocapsid into the cytoplasm during infection are two steps of the viral life cycle understudied for DENV and other flaviviruses. The viral capsid is a small highly basic protein that binds nucleic acids with low specificity. In addition, packaging signals have not been found in the viral RNA. Despite this, the viral genome is the only RNA encapsidated inside the particle. In this proposal, we will address mechanisms and define the machinery involved in dengue virus genome encapsidation and uncoating. To this end, we will combine our expertise in developing dengue virus genetic tools to dissociate overlapping functions in the viral genome together with proteomic approaches and biochemical studies. In Aim 1, we will investigate how the viral genome is freed into the cytoplasm by analyzing uncoating intermediates in infected cells and studying the fate of viral components during entry. In addition, a novel 3 dimensional single particle orbital tracking methods to trace the capsid protein during infection will be explored. In Aim 2, we will use genetic tools to dissec capsid protein requirements for particle formation and infectivity. It has been recently found that the dengue virus capsid protein suffers a variety of post-translational modification in infected cells. A comprehensive mass spectrometry analysis of capsid purified from infected cells and virions, together with the design of recombinant viruses will be used to define function of structural properties of capsid. In Aim 3, we will define the protein-protein interaction network fr dengue virus assembly and determine the function of the viral protein NS3 in this process. We will use a recently developed proteomic platform in the context of viral infections to define host components required for the assembly process. Dissecting the multiple functions and interactions of the capsid protein with host and viral components will shed light on fundamental aspects of dengue and other flavivirus replication. Importantly, the studies proposed will provide new information about viral processes still unexplored for antiviral intervention.